In higher vertebrates, nerve conduction is greatly facilitated by myelin, a lipid-rich membrane that wraps around the axon. A number of devastating demyelinating diseases threat human health, and few effective treatments exist. To develop better treatment for these diseases, we must understand the mechanisms involved in myelination. Myelin is a specialized structure with distinct lipid and protein constituents. Galactosylceramide (GalCer) and sulfatide make up approximately 30% of total myelin lipids, and more than half of these galactolipids contain fatty acid with a hydroxyl group at the C2 position (2-OH galactolipids). No other mammalian tissues contain such high concentrations of 2-OH fatty acids, suggesting that 2-OH galactolipids may play a crucial role in creating the special characteristics of myelin. Despite the extraordinary abundance of 2-OH galactolipids in myelin, there is surprisingly little understanding of the basic biochemistry and physiological role of 2-OH galactolipids. The overall goal of this study is to elucidate the pathway for myelin 2-OH lipids and their roles in myelination, myelin function, and cell signaling. A recently identified fatty acid 2-hydroxylase, FA2H, provides the precursor for the synthesis of myelin 2- OH galactolipids in oligodendrocytes and Schwann cells. FA2H and other enzymes are responsible for the increase in 2-OH very-long-chain (>C20) fatty acid contents in galactolipids during myelination. The first aim of this project is to establish the biosynthetic pathway involved in the unique lipid compositions of myelin galactolipids. Extensive biochemical analyses of FA2H will be performed to determine its physiological substrate, cofactors, and potential feedback mechanisms. Isoforms of fatty acid elongases and dihydroceramide synthases will be identified by a molecular genetic approach. More recently, it was found that reduced FA2H expression via RNAi significantly enhanced motility of D6P2T cells. Cellular 2-OH also partially blocked the upregulation of cyclin-dependent kinase inhibitors, p21 and p27, in response to a stimulus for differentiation. These observations indicate that 2-OH lipids are not only major structural components of myelin, but also function as signaling molecules to modulate cell differentiation and motility. In the second aim, the mechanism of action of 2-OH lipids in cell differentiation and motility will be determined. Transcriptional regulation for p21 and p27 will be investigated to determine the target protein modulated by 2-OH lipids, and the molecular identity of 2-OH lipid species with signaling function will be determined. The third aim is to determine the role of 2-OH galactolipids in myelin function and remyelination in adult brain. The cuprizone- induced demyelination/remyelination will be used to show FA2H is involved in remyelination. Subsequently, newly available conditional FA2H-knockout mice will be used to inactivate FA2H in adult brain. This model will be used to investigate myelin morphology, function, and remyelination in the absence of 2-OH lipids. PUBLIC HEALTH RELEVANCE To develop better treatment for devastating demyelinating diseases, we must understand the mechanisms involved in myelination. This project seeks to unravel the complex pathways for the synthesis of myelin lipids and their roles in myelin maintenance and function, as well as in cell signaling that controls proper myelination. Results obtained from this study will aid in developing better therapeutic agents for neurodegenerative diseases, such as multiple sclerosis.